Temporal and spatial heterogeneity of HER2 status in metastatic colorectal cancer

Background HER2-targeted therapies have recently emerged as an option in the management of metastatic colorectal cancer (mCRC) overexpressing HER2. However, data regarding HER2 status in primary CRC and its corresponding liver metastases are limited, potentially influencing clinical decisions. Therefore, the aim of this study was to compare the HER2 status in primary CRC and paired liver metastases. Methods Patients with mCRC who were operated from their primary colorectal cancer and their corresponding synchronous or metachronous liver metastases, in the digestive surgery department of Besançon University Hospital, between April 1999 and October 2021, were included. Tissue microarrays were constructed from matched primary CRC and liver metastastic tissue samples. HER2 status was assessed by immunohistochemistry and in situ hybridization according to Valtorta’s criteria. Results A series of 108 paired primary CRC and liver metastases, including a series of multiple liver metastases originating from the same patients (n = 24), were assessed. Among the primary CRC, 89 (82.4%), 17 (15.8%) and 2 (1.8%) cases were scored 0, 1 + and 2 + respectively. In liver metastases, 99 (91.7%), 7 (6.5%) and 2 (1.8%) were scored 0, 1 + and 2, respectively. Overall, there was a 19% discrepancy rate in HER2 status between primary CRC and metastases, which increased to 21% in cases with multiple synchronous or metachronous liver metastases in a given patient. No significant difference was found between metachronous and synchronous metastases regarding the HER2 status (p = 0.237). Conclusions Our study highlights the temporal and spatial heterogeneity of HER2 status between primary CRC and corresponding liver metastases. These findings raise the question of a sequential evaluation of the HER2 status during disease progression, to provide the most suitable treatment strategy. Supplementary Information The online version contains supplementary material available at 10.1186/s13000-024-01508-y.


Background
Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer related death worldwide with nearly 2 million new cases diagnosed and about 1 million death per year [1].Almost 50% of CRC patients will develop liver metastases and less than a third will be candidates for surgical resection [2,3].
The management of metastatic colorectal cancer (mCRC) depends on the resectability of the metastases, the patient's condition and the tumor molecular features.In many cases, several biomarkers, such as KRAS, NRAS, BRAF and MisMatch Repair (MMR) status, are routinely assessed to adapt the therapeutic strategy [4].Recently, the role of human epidermal growth factor 2 (HER2) as a new target has emerged in mCRC.HER2 is a strong oncogenic driver and trastuzumab, the first monoconal antibody blocking HER2, has become the standard treatment for HER2-positive advanced gastric cancer overexpressing HER2 [5,6].In mCRC, several phase II clinical trials have demonstrated the efficacy and tolerability of different dual HER2-targeted therapies [7][8][9][10][11].However, this clinical efficacy was optimal in patients without RAS mutations [7].More recently, a clinical trial evaluating trastuzumab conjugated to deruxtecan, a topoisomerase inhibitor, has shown promising activity in mCRC, irrespective of RAS mutation status [12,13].In these trials, patient recruitment is mainly based on immunohistochemistry and in situ hybridization.Indeed, in CRC, a specific HER2 scoring system, relying on these two techniques has been developed to provide an identification of CRC patients eligible in clinical trials [14,15].Moreover, HER2 amplification has been associated to resistance to anti-EGFR treatment in wild-type RAS and BRAF mCRC.
In this setting, it is necessary to provide an accurate assessment of HER2 status.It can be challenging in cases where tumors show a heterogeneous expression of HER2 regarding different locations.Thus, in breast cancer and gastric cancer, it has been described that these situations can lead to discrepancies in HER2 status between primary tumors and metastases [16,17].In CRC, only few studies are available regarding HER2 heterogeneity.Moreover, most of them have been based on different scoring systems, with series including various number of cases [18][19][20][21].In addition, spatial and temporal heterogeneity has never been precisely described [18][19][20][21].
Thus, the aim of this study was to compare the HER2 status between primary CRC and their corresponding liver metastases.

Patients
Patients who were operated for a primary CRC and underwent synchronous or metachronous liver metastases resection in the digestive surgery department of Besançon University Hospital, between April 1999 and October 2021, were selected for this study.

Tissue microarray manufacturing
Tissue microarrays (TMA) were constructed from the most representative primary CRC and corresponding liver metastasis formalin-fixed paraffin embedded (FFPE) blocks.The punch's diameter was 1 mm and each tumor had three TMA spots.In addition, a supplementary TMA was built from the multiple synchronous or metachronous liver metastases present in the same patient.

Determination of HER2 Status HER2 Immunohistochemistry
HER2 immunohistochemistry (IHC) was initially assessed using 4 µm sections of TMA blocks.Immunostaining was performed on the Ventana Benchmark automatic immunostainer ® (Roche diagnostics, Meylan, France), using a VENTANA anti-HER2/neu ® (4B5) rabbit monoclonal primary antibody, according to the manufacturer's instruction.In each section, there were external positive controls.
HER2 status of IHC staining was assessed according to Valtorta et al. [14].It was defined as negative (0 no staining, 1 + faint staining regardless of cellularity, 2 + moderate staining with < 50% positive cells and 3 + intense staining with ≤ 10% positive cells), equivocal (2 + moderate staining with ≥ 50% of positive cells) and positive (3 + intense staining with > 10% positive cells) and scored by two pathologists.In cases of discrepancy, consensus was reached by reviewing cases where the pathologists' interpretations initially differed.

Validation of TMA method for HER2 screening
To evaluate the reliability of the TMA method, an additional HER2 IHC on whole slides (WS) was performed for TMA spots with HER2 score of 1 + , 2 + , 3 + , as well as 10 randomly selected TMA spots IHC score of 0.

HER2 fluorescent in situ hybridization
Fluorescent in situ hybridization (FISH) was performed on WS CRC with an equivocal (2 + with ≥ 50% off positive cells) or positive (3 + with > 10% positive cells) HER2 IHC status.FISH using ZytoLight ® SPEC ERBB2/ CEN17 Dual Color Probe Kit (CliniSciences, Nanterre, France) according to the manufacturer's instruction was used to assess HER2 amplification.The scoring and evaluation were performed by counting ERBB2 and CEN17 signals from 100 non-overlapping nuclei core in tumor regions.Tumors with a ratio ERBB2/CEN17 ≥ 2 were considered amplified and otherwise were considered non-amplified [14].

Patients' characteristics
Clinical parameters were retrospectively collected by review of the medical files.These parameters included age, gender, WHO Performance Status at the diagnosis, neoadjuvant and/or adjuvant treatment, anatomical site and TNM stage according to UICC 8th edition.
The histological and molecular parameters collected included CRC histological type and grade according to the 2019 WHO Classification, lymphovascular and perineural invasion, lymph node status, MMR status and KRAS, NRAS and BRAF status.

Statistical analysis
The HER2 IHC status in the primary tumor and corresponding liver metastases were expressed as percentages with 95% confidence interval (CI) and concordance was assessed using the Cohen's kappa coefficient.The statistical analysis was performed with R software v.4.0.2.

Ethics
The project was approved by the scientific board of the Regional Biobank of Franche-Comté, France (BB-0033-00024) ensuring patients' informed consent.The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki (6th revision, 2008).

Clinicopathological characteristics
Tumor tissue samples from 108 patients who had colorectal and liver resection were collected (Fig. 1).
The relevant clinicopathological characteristics of the patients are summarized in Table 1.
A complete concordance between HER2 TMA and HER2 WS was observed in the 10 randomly selected patients with HER2 score 0.
FISH detected HER2 amplification in only one case (1/108; 0.9%) among the IHC 2+ samples, both present in the primary CRC and the corresponding liver metastasis (Fig 2).
This case corresponded to a 45 years old female patient having a low-grade NOS adenocarcinoma of the left side, associated with perforation and a synchronous liver metastasis, but without lymph node invasion.The patient was initially treated by surgery and adjuvant chemotherapy and progressed 3 years later with a pulmonary metastasis.

Concordance of HER2 status between primary tumor and liver metastasis
The overall concordance between primary CRC and their paired liver metastasis was 80.5% (Table 3).
Fig. 1 Flow chart.HER2 immunohistochemistry (IHC) was performed on the 108 paired colorectal tumor -liver metastasis.Two colorectal tumors and two liver metastases were scored 2 + and had their HER2 status assessed by fluorescent in situ hybridization (FISH).One pair (colorectal tumor and liver metastasis) was HER2 amplified and one pair was HER2 non-amplified.In addition, tumor heterogeneity was analyzed for 24 patients with multiple metastases.85 metastases were scored 0 and 8 were scored 1 + on IHC Out of 108 cases, 84 (77%), 2 (1.8%) and 1(0.92%) were respectively scored 0, 1 + , 2 + on both primary CRC and corresponding liver metastasis.For 21 patients (19%), the HER2 status of primary CRC was different from that on the liver metastasis.Five patients (4.6%) were scored 0 on primary CRC and 1 + on the liver metastasis (Fig. 2).Conversely, 14 patients (12%) showed 1 + staining on primary CRC and 0 on the liver metastasis (Fig. 2).One patient (0.92%) showed 1 + staining on primary CRC and 2 + on the liver metastasis and one patient (0.92%) showed 2 + staining on primary CRC and 0 on the liver metastasis.The Cohen's kappa coefficient was 0.17 corresponding to a very low concordance.
In patients with concordant status, 28 (32.2%) had metachronous and 59 (67.8%) synchronous metastases.Among the 21 patients who presented a discrepancy in the HER2 status between the primary CRC and the metastasis, four (19.1%) had metachronous metastasis and 17 (80.9%)had synchronous metastasis.The characteristics of these patients with discordant HER2 status are summarized in the supplementary Table 1.A chi-square test was performed and showed no significant difference between metachronous and synchronous metastases regarding the HER2 status (p = 0.237).

HER2 status in multiples liver metastases
HER2 status was analyzed for 24 patients with multiple liver metastases.The number of metastases per patient varied from 2 to 13 lesions.Overall 8 (33.3%) were scored 1 + and 16 (66.7%)were scored 0. None of the metastases was scored 2 + or 3 + .For 5 out of 24 patients, liver metastases showed a different score, leading to a discrepancy reaching 21%.It concerned 2 patients with metachronous metastases and 3 patients with synchronous metastases (Fig. 3).

Discussion
The aim of this study was to analyze the concordance of HER2 status between primary CRC and their corresponding liver metastases.Indeed, the precise evaluation of this biomarker is mandatory, as the expansion of new treatments targeting HER2 in this location has recently led to promising results, mainly in RAS wild-type tumors [7][8][9][10][11][12][13].
In our series, based on 108 patients and 285 samples, we found a significant discrepancy between primary CRC and its paired metastases reaching 19.5%.This rate reached 21% between the multiple liver metastases resected in each patient.This discrepancy concerned the  0, 1 + and 2 + IHC categories, as only one case of 2 + IHC HER2 amplified CRC was observed, with the same status on primary and metastatic sites.This low frequency of HER2 amplified CRC is in accordance with the literature's data, reporting rates between 2 and 5% [22,23].Few studies have compared the HER2 status of primary CRC and its corresponding metastases [18][19][20][21]24].Moreover, they did not use the latest recommended scoring system, as compared to our work, based on the Valtorta criteria [14].In addition, they did not analyze multiple synchronous or metachronous metastases originating from the same patient [18][19][20][21].Lee et al. reported a discrepancy rate of 14.6% between primary CRC and liver metastasis.However, the interpretation of IHC staining was based on the criteria defined for gastric cancer [18].In the study by Chen et al. discrepancy was also frequently observed in paired tumor samples encompassing primary CRC and brain metastases [24].According to the study of Shan et al., a discrepancy in liver metastases compared to primary CRC was present in 27.3% of cases [20].Recently, Hashimoto et al. found a discordance rate of 7% for HER2 amplified tumors and 19% for HER2 low tumors between primary CRC and metastases [21].Additionally, we observed a discrepancy rate reaching 21% among the multiple liver metastases resected in a given patient.This rate was similar in synchronous and metachronous liver metastases.Thus, our work highlights the temporal and spatial heterogeneity of HER2 status that can be observed in CRC.
Our study took in consideration the "HER2 low status", which includes 1 + and 2 + non-amplified cases, associated with a discrepancy rate reaching almost 19.5% between the primary CRC and its paired metastasis.This low level of HER2 expression represents an opportunity to offer a new approach with antibody-drug conjugate (ADC) such as trastuzumab deruxtecan (T-DXd) [12].This therapeutic mechanism is supported by the ADC linking to HER2 protein found on malignant cells, even with low level of expression.After internalization and cleavage, DXd causes targeted DNA damage and apoptosis in cancer cells.Thus, it is a different pathway from the targeting of HER2 2 + amplified / HER2 3 + tumors, whose aim is to neutralize the oncogenic addiction provided by HER2 overexpression.This therapeutic approach of HER2 low tumors has been successfully validated in breast cancer, is promising in gastric cancer, but has not yet demonstrated positive effects in CRC.However, in this setting, only one study is available and clinical trials regarding this approach are still ongoing [12,13,25].Therefore, this particular immunohistochemical pattern has still to be considered.
Theranostic biomarker heterogeneity remains a challenge in the management of solid tumors, potentially leading to under-or overtreatment.In this setting, many studies have been performed leading to different results according to the tumor type and the biomarker analyzed.Regarding the MMR status in CRC, the recent available studies demonstrated a high concordance rate between primary CRC and their metastases [26].However, debate Fig. 3 HER2 immunohistochemistry score among patients with multiple liver metastases.Each metastases of the 24 patients were represented according to their HER2 immunohistochemistry (IHC) score.Eight patients had metachronous metastases (orange) and 16 were diagnosed with synchronous metastases (yellow).Five patients showed a heterogeneous score surrounds the RAS and BRAF status in primary CRC and corresponding metastases.While a review regarding multiple CRC biomarkers, including RAS and BRAF status, showed a strong agreement between the primary CRC and its metastatic site(s) [27], therapeutic pressure induced by chemotherapy and/or targeted treatment may alter the status post-treatment.The CRICKET study highlights how tumors initially RAS wild-type may become resistant to anti-EGFR therapy through the emergence of RAS mutated clones, and then recover a RAS wild-type status after stopping the targeted treatment [28].These data illustrate dynamic tumor heterogeneity under treatment pressure.Taken together, these data support the use of an approach that provides a more accurate assessment of the HER2 status and overcomes heterogeneity.In this setting, liquid biopsy relying on circulating tumor DNA (ctDNA), may offer a better way to characterize HER2 status in patients with metastatic CRC.Some clinical trials, such as the TRIUMPH study, have reported a very good concordance between liquid and tissue-based approaches [10].However, this biomarker analysis was mainly designed to select HER2 amplified / 3 + tumors associated with a high level of DNA copy number, rather than to screen HER2 low tumors.As this assay is designed to detect DNA alterations, such as amplification in the blood, and not the absence or low level of protein expression represented by 0, and HER2 low CRC, which include 1 + and 2 + non amplified cases, the evaluation of HER2 by IHC remains relevant.

Conclusions
In conclusion, our study highlights the temporal and spatial heterogeneity of HER2 status between the primary colorectal tumor and synchronous or metachronous liver metastases.Our data underline a difference between HER2 low CRC, which can be taken into account in this era of precision medicine and innovative therapeutic options, and raise the question of testing different tumor sites for HER2 status.

Table 1
Clinicopathological characteristics of primary tumor from consecutive patients who underwent colorectal and liver metastasis resection for CRC

Table 2
HER2 status in colorectal tumor and liver metastasis